The present invention is based on the development of an efficient infection system for HCV, and on the finding that the human proteins annexin V, tubulin and apolipoprotein B bind to the hepatitis C virus envelope proteins E1 and/or E2 and concerns the usage of these human proteins to diagnose and treat an infection with hepatitis C virus. The present invention also relates to the usage of the latter proteins to enrich HCV envelope proteins and to molecules which inhibit binding of HCV to these human proteins, as well as vaccines employing the E1 and/or E2 binding domains.
Hepatitis C virus (HCV) infection is a major health problem in both developed and developing countries. It is estimated that about 1 to 5% of the world population is affected by the virus, amounting up to 175 million chronic infections worldwide. HCV infection appears to be the most important cause of transfusion-associated hepatitis and frequently progresses to chronic liver damage. Moreover, there is evidence implicating HCV in induction of hepatocellular carcinoma. Consequently, the demand for reliable diagnostic methods and effective therapeutic measures is high. Also sensitive and specific screening methods for HCV-contaminated blood-products and improved methods to culture HCV are needed.
HCV is a positive stranded RNA virus of about 9,8 kilobases which code for at least three structural and at least six non-structural proteins. The structural proteins have not yet been functionally assigned, but are thought to consist of a single core protein and two envelope proteins E1 and E2. The E1 protein consists of 192 amino acids and contains 5 to 6 N-glycosylation sites, depending on the HCV genotype, whereas the E2 protein consists of 363 to 370 amino acids and contains up to 11 N-glycosylation sites, depending on the HCV genotype (for review see Maertens and Stuyver, 1997). The latter envelope proteins have been produced by recombinant techniques using Escherichia coli, baculovirus, yeast and mammalian expression systems. The usage of an expression system in higher eukaryotes and especially in mammalian cell culture leads to envelope proteins of superior quality, i.e. they are effectively recognized by antibodies recovered from HCV patients (Maertens et al., 1994, de Martynoff et al., 1996).
Standardized infections of live viruses are a prerequisite for studying the binding parameters of HCV to eukaryotic cells. Controllable infection of eukaryotic cells by HCV, however, poses a problem. As a partial solution to this problem, a Daudi cell line was selected, which was supporting productive infection for HCV (Shimizu et al., 1996). However, the inocula for infection gave variable results in Molt-4 cells and even in Daudi cells. Consequently, comparative studies, e.g. on the development of drugs interfering with the interaction of HCV with its target eukaryotic cell are troublesome. Therefore, there is an urgent need for a protocol, which guarantees efficient and reliable infections of eukaryotic cells by HCV.
The HCV envelope proteins E1 and E2 interact with each other to form hetero-oligomeric complexes. Although the exact role of these HCV envelope proteins has not been elucidated yet, it has been suggested that they are responsible for binding of the virus to target cells. Indeed, binding of E2, mostly involving the highly variable amino terminus of E2 (i.e. the hyper variable region I), to target cells has been documented by several authors (Farci et al., 1996; Shimizu et al., 1994; Zibert et al., 1995 and Rosa et al., 1996). Furthermore, several host proteins have been shown to bind to one or both envelope proteins. For example, the chaperone protein calnexin has been shown to interact with both E1 and E2 and, by doing so, to support correct folding of both envelope proteins (Deleersnyder et al., 1997). Also lactoferrin, a protein mainly found in milk, has been demonstrated to bind to both envelope proteins (Yi et al., 1997b). However, the role of this interaction is unclear. A 24 kDa plasma membrane protein has been described which binds specifically to E2 (WO 97/09349 to Abrignani). The latter protein has been suggested to be a cellular receptor for HCV. Also the mannose receptor on hepatic endothelial cells and macrophages and the asialoglycoprotein receptor on hepatocytes have been suggested to function as receptors for HCV via the E1 and E2 proteins (WO92/08734 to Raiston et al.). In addition, RNA of HCV in serum of patients is often found associated with the LDL-fraction of serum (Thomsson et al., 1992 and 1993; Agnello et al., 1996 and1997). The nature of this finding, has not been unravelled yet.
Taken together, several HCV-binding proteins have been described in the literature. However, no prior art exists regarding the binding of the human proteins annexin V, apolipoproteinB, or tubulin to the hepatitis C virus envelope proteins E1 and/or E2. Neither has the interaction between HCV and LDL been further unravelled with regard to the components of LDL involved, ie protein, lipid or sugar, nor with regard to the regions of the HCV proteins involved, ie the envelope proteins E1 and/or E2.
Annexin V (also termed endonexin II, placental anticoagulant protein, PP4 or lipocortin V) is a member of the family of structurally closely related Ca2+-dependent phospholipid-binding proteins, known as annexins, which have molecular weights between 32 and 67 kDa (Klee, 1988; Zaks and Creutz, 1990). Annexin V is found in various tissues such as liver, spleen, lung, intestine and placenta (Walker et al., 1990). The protein has been described to bind, in a Ca2+-dependent manner, to placental membranes (Haigler et al., 1987) and to inhibit blood coagulation (Grundman et al., 1988) and phospholipase A2 activity in vitro (Pepinsky et al., 1988). Other investigators have demonstrated that annexin V behaves like an integral membrane protein and forms calcium-selective cation channels (Rojas et al., 1990; Bianchi et al., 1992).
We have recently shown that annexin V, present on human liver plasma membranes, specifically binds to xe2x80x9csmallxe2x80x9d HBsAg, one of the envelope glycoproteins of hepatitis B virus (HBV), in a Ca24+-dependent manner (Hertogs et al., 1993; WO 94/01554). The receptor-ligand relationship between HBsAg and annexin V is further supported by the observation that rabbits, immunized with native human liver annexin V or recombinant annexin V, or chickens, immunized with F(ab"")2-fragments of rabbit anti-annexin V IgG, spontaneously develop anti-idiotypic antibodies (Ab2) which specifically recognize HBsAg (Hertogs et al., 1994). Since HCV is an RNA virus belonging to the family of flaviviridae, and HBV is a DNA virus belonging to the family of hepadnaviruses, the binding of annexin to E1 or E2 of HCV was completely unexpected.
Tubulin is a soluble protein found in most eukaryotic cells and is the principal protein subunit of microtubules in the cell. Microtubules are the principal components of mitotic and meiotic spindles and of the axons of neuronal cells. Microtubules also participate in several aspects of intracellular transport, in maintenance of various cell surface properties such as receptor capping and they establish overall cell shape and internal cytoplasmic architecture. Tubulin extracted from neurons is a dimer of about 100 kDa. each dimer being composed of two polypeptides xcex1-tubulin (50 kDa) and xcex2-tubulin (50 kDa), which have closely related amino acid sequences (Alberts et al., 1983).
Tubulin has been implicated in the transcription of Sendai virus (Takagi et al, 1996) and has been shown to be involved during intracellular transport of herpes simplex virus type 1 (Hammonds et al., 1996) and tobacco mosaic virus (McLean et al., 1995). Tubulin appears also to interact with the matrix protein of vesicular stomatitis virus (Melki et al., 1994).
Apolipoprotein B (ApoB) represents the main protein component of the low-density lipoproteins (LDL). Besides its lipid-carrier property apoB is involved in the secretion into the plasma of newly synthesized triglyceride-rich particles and also as a ligand for the high-affinity membrane receptor responsible for the uptake and degradation of LDL (Scanu, 1987). Although E1has been implied in binding to LDL (Monazahian et al., 1995), the specific domains of E1 directly interacting have never been elucidated. Moreover, it is unclear whether the binding of E1 to LDL is mediated through the lipid or sugar component or any of the protein components of LDL.
Hence, no prior art exists which demonstrates or suggests the binding of the human proteins annexin V, tubulin or apoB to the hepatitis C virus envelope proteins E1 and/or E2 leading to the development of reliable methodological tools to diagnose HCV and effective therapeutic agents to treat or prevent HCV infections. Although annexins and tubulin have been described to interact with other viruses, there is no evidence to suppose an interaction with HCV, especially since none of the described viruses belongs to the flavivirus or pestivirus families.
It is clear from the literature that there is an urgent need to develop reliable diagnostic methods, reliable vaccines, and effective therapeutic and prophylactic agents for HCV. In addition, sensitive and specific screening methods of HCV-contaminated blood-products and improved methods to culture HCV are needed as well as reliable infection protocols. Knowing which human proteins bind to HCV, and function as putative receptor for HCV, may help in designing efficient diagnostic tools and therapeutic agents. In this regard, the present invention is based on the surprising finding that the human proteins annexin V, tubulin and apolipoprotein B bind to HCV via its envelope complex which is composed of the E1 and E2 proteins.
Therefore, the present invention aims at providing a human protein, or a functionally equivalent variant or fragment thereof, chosen from the group consisting of annexin V, tubulin and apolipoprotein B for use in the preparation of:
a composition to treat an infection with HCV, or
a method to diagnose an infection with HCV, or
a method to purify HCV proteins, or
a method for propagating HCV in cell culture.
More specifically, the present invention aims at providing a protein as defined above, wherein said protein, or a functionally equivalent variant or fragment thereof, binds to HCV, and more preferably to the envelope proteins E1 and/or E2 of HCV.
In addition, the present invention aims at providing a protein as defined above, wherein said annexin V, or a functionally equivalent variant or fragment thereof, binds to the amino acids 307-326 of E1 and/or the amino acids 413-467 of E2.
In addition, the present invention aims at providing a protein as defined above, wherein said tubulin, or a functionally equivalent variant or fragment thereof, binds to the amino acids 192-326 of E1 and/or the amino acids 384-673 of E2.
In addition, the present invention aims at providing a protein as defined above, wherein said apolipoprotein B, or a functionally equivalent variant or fragment thereof, binds to the amino acids 192-263 and/or the amino acids 288-326 of E1.
The present invention aims also at providing a composition comprising an HCV E1 and/or E2 peptide, or a functionally equivalent, or variant thereof, which contains a binding domain for a human protein as defined above, and said composition can be used prophylactically; or therapeutically. Such a composition can be used to generate antibodies interfering directly with the viral life cycle and will therefore be highly potent. Moreover, the peptide or its functionally equivalent can be used as a drug competing with the natural interaction and thus interfering directly with the viral life cycle.
It is also an aim of the present invention to provide a composition comprising a human protein as described above, or any functionally equivalent variant or fragment thereof, and a carrier for use to treat an infection with HCV. A composition which targets essential functions in the life cycle of HCV and may be highly efficient and is expected to be cross-protective against all HCV genotypes.
It should be clear that the interactions between host proteins and HCV envelope proteins may not be limited to the examples disclosed here (i.e. tubulin, annexin V and apolipoprotein B).
It is therefore an aim of the present invention to provide any host protein, or a functionally equivalent variant or fragment thereof, which interacts with HCV envelope proteins and which can be used in the preparation of a drug to treat an infection with HCV, or, a method to diagnose an infection with HCV, or, a method to purify HCV proteins, or, a method to propagate HCV in culture.
The present invention further aims at providing a method for diagnosing exposure to or infection by HCV comprising contacting HCV within a sample of body fluid with a host protein as described above, or a functionally equivalent variant or fragment thereof, and determining the binding of HCV within a sample of body fluid with a host protein as described above, or a functionally equivalent variant or fragment thereof. For example, apoB, tubulins and/or annexins may be employed to capture and/or detect HCV particles.
Moreover, the present invention aims at providing a method for purifying HCV envelope proteins comprising contacting a composition containing HCV envelope proteins with a human protein as described above, or a functionally equivalent variant or fragment thereof, and isolating the portion of the composition which binds to said protein as described above, or a functionally equivalent variant or fragment thereof.
The present invention also aims at providing an assay kit for detecting the presence of HCV comprising a solid support, a human protein as described above or a functionally equivalent variant or fragment thereof, and appropriate markers which allow to determine the complexes formed between HCV in sample of body fluid with a human protein as described above, or a functionally equivalent variant or fragment thereof.
In addition, the present invention aims at providing a method for propagating HCV in cell culture comprising providing a cell that over-expresses a human protein as described above, or a functionally equivalent variant or fragment thereof, infecting the cell with HCV, and culturing the infected cell.
Furthermore, the present invention aims at providing a method for reducing or eliminating the presence of HCV in plasma, serum, or other biological liquids which method comprises contacting said biological liquid with a human protein as described above, or a functionally equivalent variant or fragment thereof, and separating said biological liquid from said protein as described above, or a functionally equivalent variant or fragment thereof.
Moreover, the present invention aims at providing a method to determine anti-HCV antibodies in plasma, serum, or other biological liquids which method comprises allowing competitive binding between antibodies in the biological liquid and a known amount of HCV envelope protein for binding to a human protein as described above or a functionally equivalent variant or fragment thereof, and determining the amount of the HCV envelope protein bound.
The present invention also aims at providing a method to screen for molecules which modulate the binding between HCV and a human protein as described above, or a functionally equivalent variant or fragment thereof.
The present invention aims also at providing a composition comprising a molecule which modulates the binding between HCV and a human protein as described above, or a functionally equivalent variant or fragment thereof.
The present invention aims also at providing an in vitro infection method of eukaryotic cells for HCV by using purified HCV particles. More particularly, purifying HCV particles by ultra centrifugation of a solution containing said HCV particles. More particularly, purifying HCV particles by ultra centrifugation of a body fluid containing said HCV particles. More particularly, infecting Daudi, Molt, HepG2, or any B-, T-, macrophage, hepatocyte or hepatome cell line in vitro with said purified HCV particles.
The present invention aims also at characterizing said purified HCV particles for the content of E1 and/or E2 proteins. Moreover, the present invention aims also at characterizing said purified HCV particles for the content of antibodies against E1 and/or E2. Moreover, the present invention aims also at characterizing said purified HCV particles for the content of LDL. Moreover, the present invention aims also at characterizing said purified HCV particles for the content of apolipoprotein B.
The present invention aims also at providing an in vitro infection system to be used for screening for molecules which modulate the binding between HCV and eukaryotic cells.
In addition, the present invention aims at providing an in vitro infection system to be used for diagnosing exposure to or infection by HCV comprising purifying HCV particles as described above, infecting host cells as described above, and determining the multiplicity of infection.
Finally, the present invention aims at providing isolated HCV E1 and/or E2 peptides as defined by SEQ ID NO 1 to 6 (Table 4), or any fragment thereof, wherein said sequence binds to annexin V, tubulin and/or apolipoprotein B.
All the aims of the present invention are considered to have been met by the embodiments as set out below.